ИСТИНА

Sequence-specific endonucleases with extended recognition sites can cleave a unique site in complex genomes. Since these nucleases can show off-target cleavage activity, spatio-temporal control of their activity is necessary for the precise genome engineering. It would be desirable to control the enzymatic activity by an external signal, e.g. by light. Such photoregulation is based on the azobenzene "photoswitch" [1]. Azobenzene isomerizes between the extended trans- and the cis-configuration by illumination with UV (trans → cis) or blue light (cis → trans) as well as by thermal relaxation (cis → trans). We are developing the "molecular gate" strategy based on the fact that most type II restriction endonucleases (RE) are homodimers [2]. The DNA-binding center is located in the interface between the two subunits. It is possible to modify the protein at the entrance of the DNA-binding site and block its activity. To create the obstacle for DNA penetration to the active center we suggest to use the ability of oligonucleotides containing azobenzene insertion to form a duplex. Azobenzene in trans-configuration stabilizes the duplex and cis-configuration causes destabilization [3]. Thus formation and dissociation of the duplex can be reversibly photo-regulated. The strategy is illustrated for the type II RE SsoII. To choose the optimal length of the duplex 10-mer and 15-mer modified oligonucleotides were synthesized. After attachment to the protein these oligonucleotides are supposed to form 10-mer DNA duplexes. The initial rates of DNA cleavage at 37°C upon UV-illumination was 2 times higher than upon blue light illumination. Our results demonstrate the possibility of changing the enzymatic activity upon illumination.